An immune complex is an aggregate of immunoglobulins, non-immunoglobulin serum proteins, and antigens. Immune complexes are formed as a natural consequence of the immune response to antigens of infectious agents, to normal tissue components in the case of autoimmune diseases, to tumor-associated antigens, and to other antigens. The complexes are normally removed by the cells of the reticuloendothelial system. When this system is compromised or overloaded, circulating immune complexes may deposit in a number of organs, thereby causing possibly severe clinical problems. Further, in cancer, it is postulated that immune complexes may block other effector mechanisms of the immune system which would otherwise destroy malignant cells. Several studies have indicated that removal of circulating immune complexes may be an effective therapeutic technique. See, e.g., Theofilopoulos and Dixon, Adv. Immunol., 28, 90-220 (1979); Theofilopoulos and Dixon, Immunodiagnostics of Cancer, page 896 (M. Decker Inc., New York, N.Y. 1979).
Immune complexes form as a result of immunoglobulins reacting with antigens. Immunoglobulins are able to cross-link antigens so that a lattice network of immunoglobulins bound to antigens is formed. Once the antigen-immunoglobulin reaction has occurred, the immune complex can then be decorated with a variety of serum proteins such as the proteins of the complement cascade.
Complement component Clq selectively binds immune complexes in the presence of monomeric immunoglobulin because of the molecule's ability to develop a "functional affinity" when binding immune complexes. A "functional affinity" results when multiple low affinity receptors, confined in space, interact with multiple ligands, which are also confined in space. Normally, an individual ligand would rapidly associate and dissociate from the low affinity receptor but, when multiple ligands in a complex interact with multiple receptors, the dissociation from the receptors is very slow since the probability of all ligands dissociating at the same time is very low. The slower dissociation rate results in an affinity several orders of magnitude greater than the individual receptor's affinity. The difference in affinities for the individual ligand and the complexed ligand produces a selection for the complexed ligand when presented with both species.
The mature Clq molecule contains two distinct portions, the stalk and the globular head. There are six globular head regions per Clq molecule. Each contains a low affinity immunoglobulin binding site. Hughes-Jones and Gardner, Immunology, 34, 459-63 (1978); Duncan and Winter, Nature, 332, 738-40 (1988). Since there are six globular head regions on Clq, the molecule can form multiple binding interactions with the multiple immunoglobulins present in immune complexes. Id. The result is a higher net affinity for immune complexes (id.) due to the low probability of more than one bound globular head receptor dissociating simultaneously (i.e., a functional affinity develops). Thus, when Clq is presented with both immune complexes and monomeric immunoglobulin, it selectively binds to the immune complexes because of the slower dissociation kinetics of the immune complexes.
Many investigators have tried to identify the residues on immunoglobulins that are recognized by Clq. Initial theoretical studies that compared the sequences of immunoglobulin Fc regions of various species known to bind human Clq produced four possible sites in two general locations: 1) the residues flanking Trp277 and Tyr278 (residues 275-295) (Lukas et al., J. Immunol., 127, 2555-60 (1981); Prystowsky et al., Biochemistry, 20, 6349-56 (1981)); and 2) the residues flanking Glu318 (residues 316-338) (Stalinheim et al., Immunochem., 10, 501-507 (1973); Burton et al., Nature, 288, 338-44 (1980)). Various studies by authors advocating one or the other site produced conflicting results.
However, Duncan and Winter recently performed a series of more conclusive experiments. Duncan and Winter, Nature, 332, 738-40 (1988). Using recombinant DNA techniques, they were able to systematically alter the various residues of the two disputed sites. Then, by determining the ability to bind Clq of each of the resulting immunoglobulins, the actual site and specific binding residues were determined. They localized the core of the Clq interactions to residues 318, 320, and 322 in the Fc region of human IgG. Despite, the success of Duncan and Winter, the site on immunoglobulins where Clq binds may not be limited to the residues indicated by their work. In fact, other immunoglobulin residues may also be involved in the Clq-immunoglobulin interaction that could not be detected using their approach. This will not be resolved until high resolution x-ray diffraction data are obtained for the Clq-Fc region complex and the complete binding interaction is determined.
Bacterial proteins such as Staphylococcus aureus Protein A also bind to the immunoglobulin Fc region. Unlike Clq, the Protein A-immunoglobulin interaction is understood in detail. In a series of crystallographic studies by Deisenhofer, et al., the structure of human IgG Fc, Protein A, and finally the IgG Fc-Protein A Fragment B co-crystal were determined. Deisenhofer et al., Hoppe-Seyler's Z. Physiol. Chem. Bd., 359, S. 975-85 (1978); Marquart et al., J. Mol. Biol., 141, 369-91 (1980). One of the most important pieces of information to come from this structure is the exact contact residues involved in the interaction. Those residues are Met 252, Ile 253, Ser 254, Val 308, Leu 309, His 310, Gln 311, Asn 312, His 433, Asn 434, His 435, and Tyr 436 of the human IgG Fc region. These residues are located at the interface between the CH2 and CH3 regions of the Fc portion of IgG, and some of them (309-312) are in close proximity to the proposed immunoglobulin binding site for Clq (318, 320 and 322).
Unlike Clq, Protein A binds to the Fc portion of immunoglobulins with high affinity. Ellman, Arch. Biochem. Biophys., 74, 443-450 (1958). Thus, Protein A cannot differentiate between complexed and monomeric immunoglobulins.
However, PCT application WO 89/04675 teaches the preparation of analogs of Protein A that have a lower affinity for the Fc region and which can develop a functional affinity for immune complexes when arrayed in a specific manner. The analogs are analogs of a binding domain of Protein A or of related sequences from functionally similar bacterial proteins such as Protein G (see page 10). This PCT application reports that oligomers of the analogs, or an array of the analogs disposed about the surface of an insoluble matrix, develop a functional affinity for immune complexes.